Anaerobic bacterial pathogens are a serious economic burden on the agricultural industry. Bacteria of the Clostridium family represent a particular burden, because these bacteria cause serious diseases in poultry and other economically valuable domestic animals. Previous efforts to control these organisms have relied upon sanitary measures and the administration of antibiotics in the animal feed.
In particular, Clostridium perfringens (“C. perfringens”) is an anaerobic bacterium that is found in the soil, decaying organic matter, and as part of the gut flora of humans and animals. Different strains of C. perfringens are designated as biotypes A through E, depending on the spectrum of toxins produced [Justin et al., Biochemistry 41, 6253-6262 (2002); McDonel (1986) PHARMACOLOGY OF BACTERIAL TOXINS; F Dorner and J Drews (Eds.) Pergamon Press, Oxford]. Biotype A strains are of particular importance as the etiological agents of various types of gangrene and enteric diseases. A particularly serious enteric disease caused by C. perfringens is enteritis necroticans (also art-known as, “necrotic enteritis”), a gangrene of the intestines resulting in necrosis, sepsis, and hemolysis, in both humans and domesticated animals [see, Pearson et al., J. Am. Vet. Med. 188(11):1309-10 (1986); Al-Sheikhy and Truscott, Avian Dis. 21(2):256-63 (1977)]. For avians, e.g., chickens (Gallus gallus), enteritis necroticans is a significant problem. C. perfringens of either type A or type C can cause major losses, especially in production broiler chickens [Ficken and Wages, Necrotic Enteritis, In Diseases of Poultry, 10th Ed. pps 261-264 (1997)]. In addition to losses associated with necrotic enteritis outbreaks, productivity is reported to be impaired in flocks with C. perfringens-associated disease [Lovl and Kaldhusdal, Avian Pathology 30:73-81 (2001)]. As noted above, antibacterial agents inserted in the animal feed are the most common method of control. However, antibacterial agents, e.g., antibiotics, are costly and subject to increasing concerns related to the promotion of bacterial resistance.
More recently, attempts have been made to provide vaccines against harmful Clostridium species. For example, Lovland et al. [Avian Pathology 33(1):83-92 (2004)] demonstrated candidate vaccines based on C. perfringens type A and type C toxoids with an aluminum hydroxide adjuvant. Vaccination of parent hens was reported to provide specific antibodies to protect progeny against enteric lesions induced by subclinical challenge with C. perfringens. Other toxoid-based vaccines prepared from detoxified C. perfringens toxins are known [see e.g., U.S. Pat. No. 4,292,307, which describes toxoids of C. perfringens types A, B and D, Cl. oedematiens, and Cl. septicum]. 
Recombinant toxoid preparations also have been proposed. For example, Titball et al., [U.S. Pat. Nos. 5,851,827, 6,403,094, and 5,817,317] report nucleic acids that encode antigenic C. perfringens peptides, as well as the peptides themselves, and vaccines prepared from the peptides. Peptides are described for example, which have amino acid residues 261 to 300 of the natural C. perfringens alpha-toxin, but lack the phosphoplipase C and sphinogmyelin hydrolyzins domains of the natural toxin. It was further reported that these peptides induce immune protection against the natural toxin. In addition, U.S. Pat. No. 6,610,300 describes a vaccine based on an antigenic fragment of a mutein C. perfringens beta-toxin.
However, no matter whether a toxoid vaccine is derived from the native organism or is obtained recombinantly, it is considered to be economically burdensome to produce and administer toxoid proteins to animals in need of immunization, except under special circumstances (e.g., treating humans who might be allergic or sensitive to other components of a whole organism vaccine). Further, protein/toxoid based-vaccines typically require repeated booster vaccinations in order to maintain full effectiveness.
Another proposed solution has been to engineer an antigenically active virus that will produce a mutein alpha-toxin, in place of the wild-type toxin. For example, Bennett et al. [Viral Immunol. 12(2):97-105 (1999)] have demonstrated a recombinant vaccinia virus vector that expresses a nontoxic C-domain of C. perfringens alpha-toxin. Unfortunately, while several recombinant vaccinia vaccines have been proposed during the past 20 years, there are still longstanding concerns about the safety of releasing live, infectious vaccinia viruses into an environment where they might be transmitted to those people who are not resistant to this virus.
The alpha-toxin (plc gene) of C. perfringens is known to possess several biological activities including hemolytic activity, phospholipase C, sphingomyelinase, phosphodiesterase, and lethal activities. There are a number of reports in the art concerning mutations to this alpha-toxin that reduce toxicity. Schoepe, et al. [Infect. and Immun. 69(11): 7194-7196 (2001)] describe a naturally-occurring C. perfringens strain that produces a non-toxic alpha-toxin. However, it would be difficult to modify this strain to elicit immune protection against other variant, but toxic wild-type C. perfringens species.
Williamson and Titball [Vaccine 11(12):1253-1258 (1993)] showed that the region of the toxin from amino acid residues 247 to 370 alone was sufficient to immunize mice against gas gangrene experimentally induced by C. perfringens. Alape-Girón et al. [Eur. J. Biochem. 267:5191-5197 (2000)] have reported that substitutions in Asp269, Asp336, Tyr275, Tyr307, and Tyr331 reduced alpha-toxin toxicity. Nagahama, et al. [Infect. and Immun. 65:3489-3492 (1997)] reported that replacement of Asp-56, Asp-130, or Glu-152 resulted in reduced alpha-toxin toxicity. Nagahama et al. [J. Bacteriology 177:1179-1185 (1995)] reported that substitution of the histidine at position 68 with a neutral amino acid, such as glycine, in the C. perfringens alpha-toxin resulted in a complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activity of the mutein alpha-toxin. This single amino acid change was believed to inactivate one of the three zinc-binding domains of the protein. The zinc-binding domain inactivated by substitution of His68 was later denoted as Zn2 [Justin et al., Biochemistry 41:6253-6262 (2002)].
Despite the foregoing, there remains a need in the art for a safe, economical and effective method of protecting intensively cultivated domestic animals, including avians, such as chickens, from infection by Clostridium species, including C. perfringens. 
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.